ABSTRACT Fanconi anemia (FA) is an inherited bone marrow (BM) failure disorder resulting from an intrinsic defect in DNA repair leading to an increased risk of cancers such as acute myeloid leukemia and squamous cell carcinoma. Approximately 130,000 children born worldwide each year are affected by FA. Currently, the only cure for the hematologic complications of FA is an allogeneic BM or hematopoietic stem cell transplant (HSCT) from a suitable HLA donor. A key component is preparing the recipient BM using some form of conditioning to both eliminate diseased cells and promote engraftment of donor product. All of the currently used conditioning regimens for FA rely on the use of alkylating chemotherapy drugs and/or irradiation, both of which are associated with an increased risk of developing secondary malignancies, especially in DNA repair disorders like FA. As an alternative strategy, antibody drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) are a promising nongenotoxic method of facilitating engraftment of gene-modified autologous or allogeneic grafts. Recent studies have shown the effective use of ADCs with either CD45 or CD117 (c-Kit) antibodies conjugated to the immunotoxin saporin (SAP). Since the general consensus is that genotoxic conditioning should be avoided in FA and other diseases with DNA repair defects, we propose to develop novel approaches to overcome these critical limitations for current gene therapy and HSCT protocols. Thus, in Aim 1, we will develop nongenotoxic conditioning regimens for FA using a FANCA knockout mouse model to optimally deplete residual HSCs and facilitate engraftment of gene-modified or allogeneic cells. Despite eliminating as many host HSCs as safely possible, there will be a risk of remaining host HSCs, which can result in residual disease-related hematopoiesis after transplantation of gene-modified cells and also in the setting of nonmyeloablative, T-cell depleted allogeneic HSCT. Aim 2 will pursue a novel approach to eliminate residual FA cells after gene therapy or allogeneic HSCT. While Aim 1 seeks to avoid allo-HSCT complications, not all FA patients will be good candidates for gene therapy. Thus, in Aim 3, we will determine whether our novel nongenotoxic conditioning approach can deplete host HSCs and prevent host immune-mediated BM graft rejection and thus permit allogeneic HSC engraftment in Fanca-/- mice. The proposed studies will develop an entirely novel approach of nongenotoxic conditioning for autologous HSC gene therapy and as a key component of a novel regimen for allogeneic HSC transplantation. In addition, we describe an innovative strategy, applicable to both gene therapy and allogeneic transplantation, to eliminate residual and uncorrected FA hematopoietic cells that may develop into leukemic cells post-transplant.